"We had expected mutations in proteins like collagen or accessory matrix proteins to cause craniofacial malformations. Realistically, nobody suspected that these so-called 'housekeeping genes' are responsible for that sort of phenotype."

By Vanderbilt University Medical Center ,A defective link in the intracellular protein "transit system" may lie at the heart of some craniofacial defects, new research in zebrafish suggests.

In the Sept. 17 online issue of Nature Genetics, Vanderbilt University Medical Center researchers report the identification of a mutation that causes severe skeletal deformities in zebrafish by shutting down a critical protein transport pathway.

The findings are surprising, said Ela Knapik, M.D., lead investigator on the study, because this pathway is thought to be so universal that a defect would prove fatal just hours after fertilization. But the mutant fish, named crusher, hatched and survived to nine days, albeit with striking skeletal abnormalities – craniofacial defects, kinked fins and shortened body.

The pathway affected by the crusher mutation is key to transporting proteins outside of the cell. All proteins are made in the endoplasmic reticulum (ER), a labyrinthine compartment just outside the cell's nucleus. Proteins are then "packaged" into transport containers called vesicles, which traverse the gelatinous cytoplasm of the cell's interior. The vesicles eventually dock with the Golgi, a structure that resembles a pancake stack and is the last major "transit station" of the cell. In the Golgi, proteins are modified into their active, final form before being shipped out to the surface of the cell in another type of vesicle. Once they reach their destination, the proteins either empty out into the extracellular space or take up residence in the cell membrane.

"Protein transport and secretion is a fundamental function of every living cell, in every organism," said Knapik, associate professor of Medicine and Cell and Developmental Biology. Similar mutations in yeast and cultured cells were lethal from the start, suggesting that no multicellular animal would be able to survive such a defect.

But, the crusher mutation appears to only affect chondrocytes, the cells that form the fish's cartilaginous skeleton. Chondrocytes secrete proteins like collagen into the extracellular space, laying down a rigid matrix (the extracellular matrix or ECM) that will form cartilage.

Under a microscope, type II collagen can mainly be found in the extracellular space. Only small amounts can be seen in the cytoplasm.

In the crusher fish, Knapik and colleagues found no extracellular type II collagen in the mutant tissue. Instead, the protein was either stuck within a bloated ER or associated with the proteasome, the cell's garbage disposal. In addition, the Golgi appeared shrunken and abnormal. This suggested that the protein somehow missed the first leg of its journey out of the cell, getting stuck at the first transit station, the ER.

The researchers have identified the source of the defect – a gene called sec23a, which is a critical component of the vesicles that transport proteins from ER to Golgi. But since the gene is supposedly active in all cells, just why chondrocytes are the only cell type affected by the mutation remains unclear.

"The fact that it affects only chondrocytes is very strange," Knapik said.

One possibility is that the fast growth of the craniofacial skeleton, which begins forming around day three, is more sensitive to the slow-down of protein transport than other cell types. Still, the results suggest that another unidentified mechanism for protein transport may exist in the other cell types.

"We had expected mutations in proteins like collagen or accessory matrix proteins to cause craniofacial malformations. Realistically, nobody suspected that these so-called 'housekeeping genes' are responsible for that sort of phenotype."

"For me, it's fascinating that the gene we have found was the least expected."

It turns out that the zebrafish mutant has a human counterpart, making the crusher mutant the first animal model that links ER to Golgi protein transport to a human craniofacial birth defect.

In the same issue of Nature Genetics – and back-to-back with Knapik's paper – a group of researchers from the University of California at Davis report the human variant of this gene, which causes a craniofacial condition called CLSD (Cranio-Lenticulo-Sutural Dysplasia) with strikingly similar defects to the crusher fish.

Although CLSD is a rare syndrome, there are hundreds of human congenital dysmorphologies of the skeleton, some of which might involve defects in this protein trafficking pathway. Knapik's model may provide insights into these disorders.

"No craniofacial or skeletal deformities – one of the most prominent human syndromes – had ever been linked to that pathway," Knapik said. "I'm very excited that now we have an animal model to study."

Other authors on the paper include: Michael Lang, Ph.D., Lynne Lapierre, Ph.D., and James Goldenring, M.D., of Vanderbilt, and Michael Frotscher, Ph.D., of the University of Freiburg, Germany. Marshall Summar, M.D., and Todd R. Graham, Ph.D., also contributed highly valuable discussions to this research. The research was supported by the zebrafish initiative of Vanderbilt University Academic Venture Capital Fund.